Circular RNAs (circRNAs) are a class of RNAs that have been found in multiple organisms and in multiple tissues and cells and have been implicated in various disease processes and cellular pathways. Thus they represent an exciting class of molecules to study in order to better understand biological phenomena. Recent studies suggest that these molecules may bind competitively with microRNAs (miRNAs), play roles in transcriptional regulation, and are important during brain and neural development. It is contemplated that circular RNAs may be of benefit in clinical practice as biomarkers or therapeutic targets. Currently, however, the discovery of novel circular RNAs is hindered because circular RNA molecules are found in much lower amounts than linear RNA molecules. There remains a lack of standardized methods for the enrichment, sequencing, and functional analysis of circular RNA isoforms.
Multiple techniques and strategies have been used to enrich for circular RNA populations from total RNA. Ribosomal RNA depletion and exoribonuclease enzyme digestion are two of the most commonly used strategies. However, to date, no single enrichment protocol has been broadly adopted by researchers studying circular RNA. Circular RNA molecules are present in total RNA pools at 1% of mRNA levels, and their concentration is low compared to other RNA species. Thus, in order to cost effectively and efficiently interrogate the sequence of circular RNA molecules, it is advantageous to increase their concentration versus other RNAs by selectively amplifying circular RNA.
One known method of nucleic acid amplification involves synthesizing first strand cDNA molecules from RNA molecules, circularizing the first strand cDNA molecules, and replicating the circularized first strand cDNA molecules using rolling circle replication (Rolling circle amplification of RNA; U.S. Pat. No. 6,977,153). Another practice includes hybridizing primers to RNA and catalyzing synthesis of cDNA and second-strand DNA resulting in a double stranded DNA copy of a region of the RNA molecule. This double stranded DNA is then fragmented, adapter sequences are ligated to the ends and the primers corresponding to the adapter sequences are used to amplify the DNA copies of the original RNA regions. Another current practice generates cDNA and second strand DNA using a template switching mechanism (Switching Mechanism at 5′ End of RNA Template; Methods and compositions for full-length cDNA Cloning using a template-switching oligonucleotide U.S. Pat. No. 5,962,272). A template switching oligonucleotide hybridizes to the CAP site at the 5′-end of the RNA molecule and serves as a short, extended template for CAP-dependent extension of the 3′-end of the ss cDNA that is complementary to the template switching oligonucleotide. The resulting full-length single-stranded cDNA includes the complete 5′-end of the RNA molecule as well as the sequence complementary to the template switching oligonucleotide, which can then serve as a universal priming site in subsequent amplification of the cDNA. Another practice includes hybridizing primers and stopper oligonucleotides to RNA, catalyzing the synthesis of cDNA, until the elongating product nucleic acid reaches the position of an annealed oligonucleotide stopper, whereby the elongation reaction is stopped. The elongated cDNA product is then ligated to the 3′ end of the oligonucleotide stopper, thus obtaining an amplified nucleic acid portion (e.g., Nucleic Acid Transcription Method; EP Number 2,570,487).
Since circular RNA molecules share sequence homology to linear RNA, any enrichment technique that relies solely on sequence composition to enrich for circular RNA molecules will also enrich for linear RNA. In contrast, ribosomal transcript reduction strategies are routinely employed to decrease the ratio of ribosomal transcripts to other species, such as circular RNA. (Salzman, J., et al., Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PLoS One, 2012. 7(2): p. e30733; Wang, P. L., et al., Circular RNA Is Expressed across the Eukaryotic Tree of Life. PLoS One, 2014. 9(3): p. e90859; Jeck, W. R., et al., Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA, 2013. 19(2): p. 141-57; Burd, C. E., et al., Expression of linear and novel circular forms of an INK4/ARF-associated non-coding RNA correlates with atherosclerosis risk. PLoS Genet, 2010. 6(12): p. e1001233; Salzman, J., et al., Cell-type specific features of circular RNA expression. PLoS Genet, 2013. 9(9): p. e1003777). However, large amounts of RNA material must be used (20 to 60 μg of total RNA) rendering this technique impractical in most cases (Jeck, W. R., et al., Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA, 2013. 19(2): p. 141-57).
While there are a number of uses for and broadening interest in circular RNAs, these molecules have different properties than circular DNA and therefore there are some applications, treatments, and uses that are better suited to circular DNA molecules as opposed circular RNA molecules. These applications include amplification and subsequent characterization of the molecule. Current methods may generate cDNA fragments from circular RNA, however no current methods generate full cDNA copies of the circular RNA molecule, thus retaining the structure and concomitant sequence readout. This is necessary for studying the function and role of circular RNAs in disease. Thus, it is apparent that a need exists for methods to convert circular RNA molecules into DNA molecules while retaining the original circular structure.
Current methods do not specifically enrich for circular RNA species nor do they retain the circular structure of the RNA templates after cDNA synthesis, because reverse transcriptases will roll around the RNA circle and create multiple and often incomplete copies of the circular RNA template, making it impossible to identify the original circular RNA sequence after intramolecular ligation in downstream analysis. Viroids and viroid-like satellite RNAs from plants, and the human hepatitis delta virus (HDV) RNA replicate their RNA genome through an RNA-based rolling-circle mechanism catalyzed by either the nuclear RNA polymerase II or a nuclear-encoded chloroplastic RNA polymerase (Macnaughton T B, Shi S T, Modahl L E, Lai M M C. Rolling Circle Replication of Hepatitis Delta Virus RNA Is Carried Out by Two Different Cellular RNA Polymerases. Journal of Virology. 2002; 76(8):3920-3927). Neither of these practices, however, generates circular DNA directly from a circular RNA template with the goal to specifically amplify circular RNA species from a complex pool of RNA.
Thus, a need still exists for generating multiple cDNA copies from their circular RNA counterparts in order to better identify rare or previously unknown circular RNAs. In addition, since the circular RNA sequences are copied (amplified) multiple times in the cDNA, significant cost savings may be realized when assaying with next-generation sequencing machines (ex. Illumina, Pacific Biosciences) since fewer reads need to be generated for the same level of sensitivity of circular RNA detection.